Syphilis, caused by the spirochetal bacterium Treponema pallidum, continues to play prominently as a sexually transmitted disease. Syphilis also is a paradigm of bacterial chronicity and immune evasion, but virtually nothing is known about how T. pallidum carries out these enigmatic processes. Unfortunately, there is a paucity of information on the functions of T. pallidum membrane proteins that likely contribute to the spirochete's complex parasitic strategy. T. pallidum is postulated to encode 45 membrane lipoproteins (4.3% of its genome). Membrane lipoproteins typically serve many important physiological roles and also have significance as virulence factors, modular components of ABC-type transporters, stabilizers of membrane integrity, protective immune targets, and proinflammatory agonists. However, the functions of the treponemal lipoproteins have remained largely undefined. In a departure from traditional T. pallidum research, we have been crystallizing the membrane lipoproteins of T. pallidum and inferring their functions from structural determinations. State-of-the-art biophysical and biochemical techniques are being applied to corroborate functions derived from structural data. From significant progress made over the past funding interval in solving the three-dimensional structures of five T. pallidum lipoproteins (Tp32, TP0319 [TmpC; PnrA], Tp34, TP0655 [PotD], and Tp0956), it is now well documented that our structural biology approach represents a successful discovery platform for exploring lipoprotein functions in the context of T. pallidum's atypical membrane biology (a key aspect of treponemal pathogenesis). Furthermore, from prior progress, we are now able to propose hypothesis-driven experiments that will allow us to place our lipoprotein functional assignments better in the context of T. pallidum physiology and membrane biology. Given this, the Specific Aims of this renewal proposal are (1) To assess the levels of expression and membrane surface localization in T. pallidum of lipoproteins whose functions are being determined; (2) To continue to clone, express in E. coli, purify, and crystallize recombinant T. pallidum lipoproteins, with emphasis on solving their three-dimensional structures. Structural data then will be used to formulate new testable hypotheses regarding potential protein function(s); and (3) To conduct follow-up biological, biochemical, and biophysical experiments that will complement functional predictions and place them in the context of T. pallidum biology and syphilis pathogenesis. Clarifying the functions of the membrane lipoproteins is essential for understanding many of the unusual aspects of T. pallidum membrane biology and its relationship to syphilis pathogenesis.